Lacquer tree (Toxicodendron vernicifluum) is an important economic crop and is rich in flavonoids. ACC (1-aminocyclopropane-1-carboxylic acid) is the precursor to ethylene. ACC treatment can induce physiological and biochemical responses in plants. In this study, flavonoids in the leaves of Toxicodendron vernicifluum, treated with ACC at different time points, were identified and analyzed. A total of 83 flavonoids were identified, including 38 flavonoids and 45 flavonoid glycosides. Among these, 48 flavonoids were first reported in T. vernicifluum. The total flavonoid content significantly increased following ACC treatment, although the accumulation patterns of individual flavonoids varied. Flavonoids with similar structure or glycosylation/acetylation modifications exhibited a positive correlation in their content changes in T. vernicifluum leaves under ACC treatment. Transcriptome sequencing was conducted on control and 3-days ACC-treated leaves, revealing an upregulation in the expression of genes related to flavonoids biosynthesis, such as PAL, CCR, CHS, MYB and ERF encoding genes. We hypothesized that ACC regulated flavonoids biosynthesis by activating ERF and MYB transcription factors in the ethylene signaling pathway. This study provided evidence for the regulation of flavonoids biosynthesis in lacquer trees through ACC treatment.